The two main protocols for this project have been approved and are in active recruitment and accrual stages now. We continue to enroll a cohort of patients with severe atopic dermatitis (AD) alone, and those with AD in the context of immune deficiency. We have a substantial cohort of patients with genetic disorders, which include atopy as a part of the syndrome (now numbering over 200 patients total). Four main findings have occurred in this project within the past year, describted in detail below. Finding 1: Because of our interest in patients with symptoms of atopy in the context of unidentified syndromes, we have been studying a series of families with cold urticaria, antibody deficiency, autoimmunity and granulomatous disease segregating in an autosomal dominant pattern. We have named this syndrome PLAID, as described in our report in the New England Journal of Medicine. Patients have low IgM and IgA, low NK cells, normal T cells and low to absent class-switched B cells. In collaboration with Dr. Daniel Kastner's lab, we found that the disease is caused by genomic deletions in PLCG2. The deletions are in an autoregulatory domain, leading to gain of PLCG2 enzymatic function but loss of distal signaling function, resulting in defective calcium flux and ERK phosphorylation in response to surface receptor cross-linking in B and NK cells, poor in vitro expansion and switched immunoglobulin secretion in response to SAC and CpG, and abnormal B-cell receptor editing in B-cells. We have found that the cold spontaneously activates certain cells in these patients, such as B cells and mast cells. Neutrophils from these patients appear to be constitutively active, perhaps explaining the granulomatous disease seen in some of the patients. In addition, again in collaboration with the Kastner lab, we have participated in the identification and cellular study of another family with an autoinflammatory disorder affecting lungs, skin and eyes, as well as loss of class switched B cells. This family has heterozygous point mutations in the same SH2 domain of PLCg2, which is deleted in PLAID. We have termed this disease APLAID, to reflect the autoinflammatory component. We have characterized major differences between PLAID and APLAID patients, including the increased cellular responses to ligand and lack of cold response in APLAID patients. Finding 2: We have been able to study the role of STAT3 in lymphocyte homeostasis and atopy. As reported last year, and now published in Immunity, we found that HIES patients have a specific defect in central memory T cells, which appears to predispose them to chronic virus reactivation, such as shingles and EBV. In addition, in order to assess the effects of STAT3 mutations on allergic disease, we performed detailed allergic histories on the majority of the NIH AD-HIES cohort and compared them to patients with marked IgE elevations but no STAT3 mutation. Patients with HIES had a substantially lower incidence of food allergy, asthma and anaphylaxis compared to those with similarly elevated IgE levels. Allergen-specific IgE was measured using ImmunoCap RAST and prominent allergen-specific IgE was measured in most patients, arguing against a defect in antibody specificity. Foxp3+ regulatory T cells (Tregs) have been shown to directly inhibit mast cell function, and many models of experimental atopy can be suppressed with regulatory T cells. We enumerated Foxp3+ T cells in HIES patients and find that within the diminished central memory compartment, there is a marked enrichment of Tregs compared to control. We have also shown that the defect appears primarily to be in mast cells, as STAT3 knockdown of human mast cells leads to marked reduction in the capacity for degranulation. Using a mouse model of AD-HIES in collaboration with Juan Rivera and John O'Shea's labs, we found that systemic IgE crosslinking or addition of mast cell secretagogue leads to impaired anaphylaxis in AD-HIES mice compared to controls. We have therefore identified STAT3 as a critical mediator of acute mast cell degranulation via the study of allergic disease in this rare immune deficiency. This report is currently under revision in a major immunology journal. Finding 3: Wet-wrap therapy is a mainstay of care for our patients with severe atopic dermatitis regardless of underlying etiology. We have now performed this therapy on over 30 patients refractory to the outpatient standard of care. We have found that while there is a transient drop in AM cortisol, this recovers, and there are few short or long term adverse affects of this therapy. We also found that there is an acute drop in circulating eosinophils, but this too recovers over time. Despite that, the long-term improvement in objective rash burden as well as subjective quality of life measures is quite significant and never before observed for such a prolonged follow up period in such a diverse patient group. Finding 4: In our study of patients with immune deficiencies and atopic phenotypes, we have developed a flow-based assay for assessing multiple STAT responses to individual cytokines. Using this tool, in collaboration with Dr. Steve Holland's lab, we have found a family with invasive cryptosporidial infections, bronchiectasis and antibody deficiency with marked elevations in serum IgE. We found absent IL-21 signaling in these patients. In collaboration with Christoph Klein in Munich, we found a 6 bp in-frame deletion in the IL-21 receptor in these patients. Dr. Klein had followed two patients with similar phenotypes and found an independent mutation in the IL-21 receptor. The report of ours and Dr. Klein's patients is under final review. In addition, we have used this tool to identify patients with gain of function mutations in STAT1 from the Holland lab and in collaboration with Neil Romberg at Yale, which lead to a variety of clinical phenotypes, and we have made inroads into the pathogenesis of these mutations leading to the discrete phenotypes. Three different manuscripts describing these findings are under review in a major Allergy/Immunology journal.